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Published on: July 1, 2013
[Purification and identification of pregnancy associated plasma protein A]
Zhao-wei Zeng1, Jing Yang, Xue-qian Wang
1Graduate School of Tianjin Medical University, Tianjin 300070, China. zengzhaowei-1@163.com
Aim:
To purify pregnancy associated plasma protein A (PAPP-A) from the pregnancy serum using the methods of G200 gel filtration, reverse affinity chromatography and DEAE ion exchange.
Methods:
The male sera were purified by G200 gel filtration and injected into rabbits to obtain rabbit-anti-human antibodies. The antibodies were linked to CNBr-activated G25 gel to make the reverse affinity chromatography. The full-term pregnancy sera were collected and centrifuged, followed by G200 gel filtration, reverse affinity chromatography and DEAE ion exchange. ELISA and SDS-PAGE were performed to detect the activity and the purity of PAPP-A.
Results:
As detected by the double diffusion, the rabbit-anti-human antibodies were active in the dilution of 1:16, and were further used for purification of PAPP-A. The PAPP-A exited in the last peak with the pH3.0 elutriant by SPA, the first peak by G200 gel filtration, the peak with 0.1 mol/L PBS elutriant by the reverse affinity chromatography, and the peak with 0.45 mol/L NaCl elutriant by the DEAE ion exchange. The SDS-PAGE results indicated that the triple treatments of the pregnancy sera by G200 gel filtration, the reverse affinity chromatography and the DEAE ion exchange effectively enhanced purity of PAPP-A, with a single protein band shown by Western blot, whereas a single purification method produced dirty bands.
Conclusion:
High purity PAPP-A has been obtained, ready for preparation of its monoclonal antibody. The study paves a way for development of PAPP-A based ELISA kits for clinical use.
